With advancing base sequence analysis technologies such as DNA sequencers, complete genome sequences have been analyzed in a variety of biological species such as the human. These analyzed genome sequences belong to specific individuals, and studies on differences in genome sequence among individuals have been launched as the next stage. However, it is difficult to analyze complete genome sequences of respective individuals, because it takes enormous time and cost for the current base sequence analysis technologies to analyze complete genome sequences in an individual-to-individual manner. Demands have therefore been made to provide a base sequence analysis technology that can analyze genome sequences in a short time at low cost. Specifically, DNA sequencers desirably have higher throughputs and analyze samples in smaller amounts.
For enabling DNA sequencers to have higher throughputs, M. Margulies et al. discloses a process using bead handling and pyrosequencing technologies in combination in Nature 437, 376-380 (2005). According to this process, DNA to be analyzed is immobilized to beads, and 45×104 beads are integrated (packed) and concurrently subjected to pyrosequencing. Thus, a base sequencing rate per one base can be increased. The pyrosequencing is a base sequence analysis technology utilizing that pyrophosphoric acid is released when a deoxynucleotide triphosphate (dNTP) is taken into a double-stranded DNA as a result of a synthesis reaction (extension reaction) of the double-stranded DNA catalyzed by a DNA polymerase. A DNA (sample DNA) whose base sequence is to be analyzed is hybridized with a DNA (primer DNA) deciding the start point (origin) of complementary strand synthesis. Four dNTPs (dATPαS, dTTP, dGTP, and dCTP) are sequentially fed to the hybridized DNA in the presence of a DNA polymerase, and, when an extension reaction occurs, pyrophosphoric acid is formed in a number corresponding to the number of extended bases. The formed pyrophosphoric acid is converted to light emission by an enzymatic reaction by the catalysis of luciferase, and the light emission is detected with a photoelectric transducer. The base sequence of the sample DNA can be determined by detecting the presence or absence of an extension reaction and/or determining the quantity of light emission when different dNTPs are added. In this process, dATPαS is generally used as a dNTP instead of dATP, because dATP and ATP are resemble with each other in structure, and background light is not trivial when dATP is used as the dNTP.
On the other hand, electrical DNA sequencings and single nucleotide polymorphism (SNP) typings have been conducted using, for example, field-effect transistor (FET) sensors and pH sensors instead of using optical detection techniques. A DNA sequencer using FET sensors detects an extension reaction by the catalysis of a DNA polymerase as a variation in surface potential (T. Sakata et. al., Angew. Chem. Int. Ed. 45, 2225-2228 (2006)). Specifically, a sample DNA is hybridized with a primer DNA that has been immobilized to a surface of a FET sensor, and four dNTPs (dATPαS, dTTP, dGTP, and dCTP) are sequentially fed thereto in the presence of a DNA polymerase. When an extension reaction occurs, a surface potential of the FET sensor decreases, because phosphates groups in side chain of the DNA have negative charges in an aqueous solution, and when the number of bases of the DNA immobilized to the surface of the FET sensor increases, the amount of negative charges on the surface of the FET sensor increases. By detecting the variation in surface potential using the FET sensor, the base sequence of the sample DNA can be determined.
In a pyrophosphate-detecting sensor using a pH sensor (Japanese Patent No. 3761569), pyrophosphoric acid formed as a result of a nucleic-acid amplification reaction, such as a polymerase chain reaction (PCR), is enzymatically converted into a hydrogen ion, and the increased hydrogen ion is detected with the pH sensor. Whether a polymerase chain reaction (PCR) is detected from how much the pH varies, and a single nucleotide polymorphism (SNP) of a sample DNA is determined.